Engineering a peptide epitope display system on filamentous bacteriophage

Abstract: The genome of bacteriophage fd has been engineered to allow foreign amino acid sequences to be displayed in the exposed N-terminal segment of the major coat protein in the virus particle: small peptides can be encoded directly; larger peptides are encoded in hybrid virions, in which wild-type coat protein subunits are interspersed with coat proteins displaying the foreign peptides. Biophysical techniques, such as X-ray diffraction, indicate that the inclusion of the peptides can be achieved without significant disturbance to the helical parameters that define the protein—protein interactions in the assembled virion and the exposure of the peptides can be verified by analysing the susceptibility to attack by proteolytic enzymes. Peptide sequences from the V3 loop of the surface glycoprotein gp120 of HIV-1 strain MN (HIV-1_MN) displayed in this way are remarkably effective structural mimics of the natural epitope. They are recognised by human HIV antisera and evoke high titres of virus-neutralizing antibodies in mice. Antibody production is stimulated by simultaneous inoculation with T cell epitopes similarly displayed on filamentous bacteriophage. The bacteriophage display system offers a powerful means of studying the immunological recognition of proteins. The specificity of the immune response, the ability to recruit helper T cells, the lack of need for external adjuvants and the structural mimicry of defined peptide epitopes, suggest that it will also be an inexpensive and simple route to the production of effective vaccines.     

New vectors for peptide display on the surface of filamentous bacteriophage

We have modified the genome of the filamentous bacteriophage fd and also constructed a number of new vectors for the purpose of displaying peptides on the surface of the virion. These vectors facilitate the directional cloning of DNA encoding a peptide of interest at or near the N terminus of the major coat protein, the product of the bacteriophage gene VIII, and the construction of hybrid capsids in which the modified coat protein is interspersed with wild-type coat protein subunits.     

Rapid Preparation of Stable Isotope Labeled Peptides that Bind to Target Proteins by a Phage Library System

We have developed a system for directly isolating foreign peptides displayed on the N-terminus of the major coat protein of bacteriophage M13. The phage particle in this system is formed as a mixture of wild type and modified coat proteins. The N-terminal segment of the modified coat protein was mutated for chemical cleavage, in order to obtain the displayed peptide from the major coat protein. Using ¹³C, ¹⁵N- labeled medium, we introduced stable isotopes, ¹³C and/or ¹⁵N, into the coat proteins. The NMR spectra for the cleaved peptides from the phage particles could be recorded within a few days after the selection of the phage clone.     

Dual genetically encoded phage-displayed ligands

M13 bacteriophage display presents polypeptides as fusions to phage coat proteins. Such phage-displayed ligands offer useful reagents for biosensors. Here, we report a modified phage propagation protocol for the consistent and robust display of two different genetically encoded ligands on the major coat protein, P8. The results demonstrate that the phage surface reaches a saturation point for maximum peptide display.     

A simple method for displaying recalcitrant proteins on the surface of bacteriophage lambda

Bacteriophage lambda (λ) permits the display of many foreign peptides and proteins on the gpD major coat protein. However, some recombinant derivatives of gpD are incompatible with the assembly of stable phage particles. This presents a limitation to current λ display systems. Here we describe a novel, plasmid-based expression system in which gpD deficient λ lysogens can be co-complemented with both wild-type and recombinant forms of gpD. This dual expression system permits the generation of mosaic phage particles that contain otherwise recalcitrant recombinant gpD fusion proteins. Overall, this improved gpD display system is expected to permit the expression of a wide variety of peptides and proteins on the surface of bacteriophage λ and to facilitate the use of modified λ phage vectors in mammalian gene transfer applications.     

Nonuniform size distribution of nascent peptides. The effect of messenger RNA structure upon the rate of translation.

The size distribution of bacteriophage MS2 coat protein nascent chains purified from MS2-infected Escherichia coli has been determined. Accumulations of nascent chains of discrete sizes were observed, providing evidence that the rate of chain elongation during coat protein biosynthesis is not uniform. A correlation of the size of nascent peptides which accumulate during MS2 coat protein biosynthesis and the position on the MS2 coat protein mRNA of the ribosome carrying those lengths of nascent peptides may be made. This correlation leads to the hypothesis that regions of mRNA secondary structure impede the movement of ribosomes during chain elongation and thus serve as the origin of the accumulation of nascent chains of discrete sizes.     

Design, construction and function of a multicopy display vector using fusions to the major coat protein of bacteriophage M13.

Incorporation of numerous copies of a heterologous protein (bovine pancreatic trypsin inhibitor; BPTI) fused to the mature major coat protein (gene VIII product; VIII) of bacteriophage M13 has been demonstrated. Optimization of the promoter, signal peptide and host bacterial strain allowed for the construction of a working vector consisting of the M13 genome, into which was cloned a synthetic gene composed of a lac (or tac) promoter, and sequences encoding the bacterial alkaline phosphatase signal peptide, mature BPTI and the mature coat protein. Processing of the BPTI-VIII fusion protein and its incorporation into the bacteriophage were found to be maximal in a host bacterial strain containing a prlA/secY mutation. Functional protein is displayed on the surface of M13 phage, as judged by specific interactions with antiserum, anhydrotrypsin, and trypsin. Such display vectors can be used for epitope mapping, production of artificial vaccines and the screening of diverse libraries of proteins or peptides having affinity for a chosen ligand. The VIII display phage system has practical advantages over the III display phage system in that many more copies of the fusion protein can be displayed per phage particle and the presence of the VII fusion protein has little or no effect on the infectivity of the resulting bacteriophage.     

The energetics of consensus promoter opening by T7 RNA polymerase

The 50-residue major coat protein (MCP) of Ff bacteriophage exists as a single-spanning membrane protein in the Escherichia coli host inner membrane prior to assembly into lipid-free virions. Here, the molecular bases for the specificity and stoichiometry that govern the protein-protein interactions of MCP in the host membrane are investigated in detergent micelles. To address these structural issues, as well as to circumvent viability requirements in mutants of the intact protein, peptides corresponding to the effective alpha-helical TM segment of wild-type and mutant bacteriophage MCPs were synthesized. Fluorescence resonance energy transfer (FRET) experiments on the dansyl and dabcyl-labeled MCP TM domain peptides in detergent micelles demonstrated that the peptides specifically associate into non-covalent homodimers, as postulated for the biologically relevant membrane-embedded MCP oligomer. MCP peptides labeled with short-range pyrene fluorophores at the N terminus displayed excimer fluorescence consistent with homodimerization occurring in a parallel fashion. Variant peptides synthesized with single substitutions at helix-interactive positions displayed a wide range of dimer/monomer ratios on SDS-PAGE gels, which are interpreted in terms of steric volume, presence or absence of beta-branching, and the effect of polar substituents. The overall results indicate discrete roles for helix-helix interfacial residues as packing recognition elements in the membrane-inserted state, and suggest a possible correlation between phage viability and efficacy of MCP TM-TM interactions.     

Conserved residues of the leader peptide are essential for cleavage by leader peptidase

Gene 8 of bacteriophage M13 codes for procoat, the precursor of its major coat protein. Gene 8 has been cloned into a plasmid and mutagenized. We have isolated mutants of this gene in which procoat is synthesized but is not processed to coat protein. We now describe mutants in the leader region of procoat, at positions -6, -3, and -1 with respect to the leader peptidase cleavage site. These positions are quite conserved among the leader peptides of various pre-proteins. Each of these mutant procoats is synthesized at a normal rate and inserts correctly into the plasma membrane, as judged by its accessibility to protease in intact spheroplasts. Procoat accumulates, largely in its transmembrane form, and is not cleaved to coat. In detergent extracts, the mutant procoats are very poor substrates for added leader peptidase. We conclude that these 3 residues are not conserved for insertion across the membrane but are part of an essential recognition site for the leader peptidase.     

Regulation of filamentous bacteriophage length by modification of electrostatic interactions between coat protein and DNA

Bacteriophage fd gene VIII, which encodes the major capsid protein, was mutated to convert the serine residue at position 47 to a lysine residue (S47K), thereby increasing the number of positively charged residues in the C-terminal region of the protein from four to five. The S47K coat protein underwent correct membrane insertion and processing but could not encapsidate the viral DNA, nor was it incorporated detectably with wild-type coat proteins into hybrid bacteriophage particles. However, hybrid virions could be constructed from the S47K coat protein and a second mutant coat protein, K48Q, the latter containing only three lysine residues in its C-terminal region. K48Q phage particles are approximately 35% longer than wild-type. Introducing the S47K protein shortened these particles, the S47K/K48Q hybrids exhibiting a range of lengths between those of K48Q and wild-type. These results indicate that filamentous bacteriophage length (and the DNA packaging underlying it) are regulated by unusually flexible electrostatic interactions between the C-terminal domain of the coat protein and the DNA. They strongly suggest that wild-type bacteriophage fd makes optimal use of the minimum number of coat protein subunits to package the DNA compactly.     

Properties of the ribonucleic acid bacteriophage ZIK/1 coat protein and its synthesis in an Escherichia coli cell-free system

The coat protein subunit of the RNA bacteriophage ZIK/1 has a molecular weight of 12100 and does not contain histidine, methionine and cysteine. The amino acid composition of the coat protein is different from that of other RNA bacteriophage coat proteins. Bacteriophage ZIK/1 belongs to a class of RNA bacteriophages distinct from the f2 type, which lack histidine in their coat proteins, and the Qβ type, which lack histidine and methionine. Bacteriophage ZIK/1 RNA is an efficient template in the Escherichia coli cell-free system producing coat protein as the major product and a number of non-coat proteins. This result is similar to that obtained with RNA from f2-type bacteriophages. It is probable that the genomes of RNA bacteriophages are structurally similar and that differences between the types of RNA bacteriophage arise from minor differences in RNA sequence.     

The primary structure of the coat protein of the broad-host-range RNA bacteriophage PRR1.

The complete amino acid sequence of the coat protein of RNA bacteriophage PRR1 is presented. After thermolysin digestion, 26 peptides were isolated, covering the complete coat protein chain. Their alignment was established in part using automated Edman degradation on the intact protein, in part with overlapping peptides obtained by enzymic hydrolysis with trypsin, pepsin, subtilisin and Staphylococcus aureus protease, and by chemical cleavage with cyanogen bromide and N-bromosuccinimide. To obtain the final overlaps, a highly hydrophobic, insoluble tryptic peptide was sequenced for seven steps by the currently used manual dansyl-Edman degradation procedure, which was slightly modified for application on insoluble peptides. PRR1 coat protein contains 131 amino acids, corresponding to a molecular weight of 14534. It is highly hydrophobic, and the residues with ionizable side chains are distributed unevenly: acidic residues are absent in the middle third of the sequence, whereas a clustering of basic residues occurs between positions 44 and 62. PRR1 coat protein was compared with the coat proteins of RNA coliphages MS2 and Q beta, and the minimum mutation distance was calculated for both comparisons. It is highly probable that PRR1. Q beta and MS2 share a common ancestor. The basic region present in the three coat proteins is recognized as an essential structural feature of RNA phage coat proteins.     

Homodimeric peptides displayed by the major coat protein of filamentous phage

Peptide libraries displayed by filamentous bacteriophage have proven a powerful tool for the discovery of novel peptide agonists, antagonists and epitope mimics. Most phage-displayed peptides are fused to the N terminus of either the minor coat protein, pIII, or the major coat protein, pVIII. We report here that peptides containing cysteine residues, displayed as N-terminal fusions to pVIII, can form disulfide-bridged homodimers on the phage coat. Phage clones were randomly selected from libraries containing one or two fixed Cys residues, and surveyed for the presence of peptide-pVIII homodimers by SDS-PAGE analysis that involved pretreatment of the phage with reducing or thiol-modifying agents. For all phage whose recombinant peptide contained a single Cys residue, a significant fraction of the peptide-pVIII molecules were displayed as dimers on the phage coat. The dimeric form was in greater abundance than the monomer in almost all cases in which both forms could be reliably observed. Occasionally, peptides containing two Cys residues also formed dimers. These results indicate that, for a given pVIII-displayed peptide bearing a single Cys residue, a significant fraction of the peptide (>40 %) will dimerize regardless of its sequence; however, sequence constraints probably determine whether all of the peptide will dimerize. Similarly, only occasionally do peptides bearing two Cys residues form intermolecular disulfide bridges instead of intramolecular ones; this indicates that sequence constraints may also determine dimerization versus cyclization. Sucrose-gradient analysis of membranes from cells expressing pVIII fused to a peptide containing a single Cys residue showed that dimeric pVIII is present in the cell prior to its assembly onto phage. A model of the peptide-pVIII homodimer is discussed in light of existing models of the structure and assembly of the phage coat. The unique secondary structures created by the covalent association of peptides on the phage surface suggest a role for homo- and heterodimeric peptide libraries as novel sources of bioactive peptides.     

Structural basis of the temperature transition of Pf1 bacteriophage

The filamentous bacteriophage Pf1 undergoes a reversible temperature-dependent transition that is also influenced by salt concentrations. This structural responsiveness may be a manifestation of the important biological property of flexibility, which is necessary for long, thin filamentous assemblies as a protection against shear forces. To investigate structural changes in the major coat protein, one- and two-dimensional solid-state NMR spectra of concentrated solutions of Pf1 bacteriophage were acquired, and the structure of the coat protein determined at 0 degrees C was compared with the structure previously determined at 30 degrees C. Despite dramatic differences in the NMR spectra, the overall change in the coat protein structure is small. Changes in the orientation of the C-terminal helical segment and the conformation of the first five residues at the N-terminus are apparent. These results are consistent with prior studies by X-ray fiber diffraction and other biophysical methods.     

Cleavage of the N-terminal formylmethionine residue from a bacteriophage coat protein in vitro

Studies were made of the N-terminal formylmethionine content of nascent and complete coat protein of bacteriophage Qβ synthesized in an Escherichia coli cell-free system. Under normal conditions of cell-free protein synthesis the formylmethionine residue was retained by all the nascent chains but by only about 50% of the completed coat protein molecules. If 2-mercaptoethanol was omitted from the cell-free system, the formylmethionine residue was cleaved during the course of peptide chain elongation. All nascent peptides which contained fewer than 40±5 amino acids retained the formylmethionine residue. Thereafter, the proportion of nascent peptides lacking the residue increased with peptide length to about 70% for nearly full length nascent peptides and complete released coat protein molecules.     

Gene overlap results in a viral protein having an RNA binding domain and a major coat protein domain

L-A double-stranded RNA (dsRNA) replicates in vivo in yeast in a conservative, asynchronous (first [+] strand then [-] strand), intraviral process. New particles are formed by packaging (+) strands. Added viral (+) single-stranded RNA (ssRNA) is specifically bound by empty virus-like particles (VLPs) and, in a reaction requiring a host factor, is converted in vitro to dsRNA. We find that the isolated binding complex replicates only if it was formed in the presence of the host factor. The VLP minor 180 kd protein, but not the major coat protein, has ssRNA binding activity on Western blots. The 180 kd protein shares a common antigenic domain with the major coat protein, the latter known to be encoded by L-A dsRNA. The 180 kd protein, but not the major coat protein, also shares an antigenic domain with a sequence encoded by the 3' end of the L-A (+) strand. Thus the 180 kd protein is also encoded by L-A dsRNA and consists of a major coat protein domain and a ssRNA binding domain.     

Missense misreading of asparagine codons as a function of codon identity and context

During asparagine starvation the frequency of lysine for asparagine substitutions increases to levels that enable one to isolate and sequence mistranslated protein. We have used site-directed mutagenesis to construct a series of derivatives of the gene encoding the coat protein of the bacteriophage MS2. The mutant set constructed has either AAU or AAC as codon three in the gene with each possible adjoining 3' base. Lysine incorporation in coat protein encoded by these genes shows that AAU is misread from 4- to 9-fold more frequently than AAC with any 3' context. Although in some cases context effects of approximately 2-fold were noted, there seems to be no simple hypothesis to explain them.     

Mutual influence of the secondary structure and information content of a messenger RNA

The presence of secondary structure in a messenger RNA suggests that natural selection has moulded the nucleotide sequence to meet the demands of base-pairing as well as those of the encoded amino acid sequence. Despite the degeneracy of the genetic code, neither requirement can be fulfilled independently of the other, so the evolved RNA and protein structures must represent a compromise. One feature of this compromise is illustrated by the structure of the coat protein gene of bacteriophage MS2. Those amino acid residues which, when represented by base-paired codons, impose the most severe limitations on the overall protein sequence, show a clear tendency to avoid being encoded in base-paired regions of the messenger RNA.